Customized oligonucleotide microchips that convert multiple genetic information to simpler patterns, are portable and reusable

ABSTRACT

This invention relates to using customize oligonucleotide microchips as biosensors for the detection and identification of nucleic acids specific for different genes, organisms and/or individuals in the environment, in food and in biological samples. The microchips are designed to convert multiple bits of genetic information into simpler patterns of signals that are interpreted as a unit. Because of an improved method of hybridizing oligonucleotides from samples to microchips, microchips are reusable to transportable. For field study, portable laser or bar code scanners are suitable.

CROSS-REFERENCE INFORMATION

This application is a Divisional of copending U.S. application Ser. No.10/212,476, filed Aug. 5, 2002, which is a Divisional of U.S.application Ser. No. 09/261,115, filed on Mar. 3, 1999, now U.S. Pat.No. 6,458,584, which is a Continuation-In-Part of U.S. application Ser.No. 08/780,026, filed on Dec. 23, 1996, now abandoned.

A novel microchip is customized to answer specific questions and hasoligonucleotides positioned on the microchip so that multiple bits ofinformation are evidenced to a simpler pattern A new method ofhybridization to a microchip is also presented.

GOVERNMENT INTERESTS

The U.S. government has rights to the invention pursuant to contractW-31-109-ENG between the U.S. Department of Energy (DOE) and theUniversity of Chicago representing Argonne National Laboratory.

BACKGROUND

Differences in nucleotide and amino acid sequences may be exploited toanalyze environmental, food or biological samples. Detection andidentification of microorganisms is important for clinical purposes andfor determination of contaminated food, air, water or soil. Studies inenvironmental microbiology are often limited by the inability tounambiguously identify and directly quantify the enormous diversity ofnatural populations. This problem is now changing with increasing use ofmolecular techniques to directly measure different genetic features.(Mobarry et al., 1996; Stahl, 1995; Wagner et al., 1995) For example,DNA probes are now commonly used to detect by hybridization, genesencoding proteins involved in specific catabolic functions, and toresolve different genetic populations in the environment. In particular,the use of group-specific DNA probes complementary to the small subunit(SSU) 16S rRNA has provided a comprehensive framework for studies ofmicrobial population structure in complex systems. Sequencing of thissubunit revolutionized microbial classification and led to the discoveryof archebacteria. (Woese, 1987) A large number of the sequences fordifferent organisms has been collected. (Maidak et al., 1996) Everymicroorganism species is characterized by a specific DNA sequence withina variable region of its ribosomal RNA gene or other genes. A highlyefficient procedure for microorganism classification and forconstruction of their evolutionary trees is based on these observations.Identification of specific sequences in ribosomal DNA is a reliablemicrobial analysis that can be carried out by direct DNA sequencing.However DNA sequencing is a rather complicated, expensive and timeconsuming procedure to use for serial microbial analysis on a commercialscale for environmental or medical applications. Consequently, newmethods are needed to make sequence matching commercially feasible.

Also, methods are needed that are transportable to the field. A nucleicacid hybridization is a highly specific and sensitive procedure thatallows a specific sequence to be detected and identified among othermillions of sequences in a genome of higher organisms, or among amixture of different organisms. The principle of hybridization is thatsequences hybridize as a function of the similarity of their linearnucleotide sequence. The hybridization of DNA or RNA extracted fromevien a very complicated mixture to a specific oligonucleotide probe hasresulted in unambiguous identification of specific microorganisms in anenvironmental sample, for example. In the course of such an analysis,RNA or DNA is extracted from a sample of microorganisms isolated fromwater solutions, air or soil, immobilized on a filter and thenhybridized successively with several oligonucleotide probes fordifferent microorganisms. However, for this purpose, the sample needs tobe checked for the presence of hundreds or thousands of differentoligonucleotides corresponding to various microorganisms which isprohibitively laborious and expensive using present methods and yieldsresults that must be interpreted by a computer in order to decipher theidentification. What is needed is a simplified pattern to provide rapidanswers to specific questions, e.g. are any known pathogens in a watersample?

The scope of applications of nucleotide hybridization is often limitedby the nature of the assays, generally involving the independenthybridization and interpretation of multiple environmental samples tomultiple DNA probes. In addition, some detection assays requireamplification of the target nucleic acid, for example, via PCR. This maycontribute to quantitative biases. Thus, there is need for assays thatprovide for greater sample through-put capacity and greater sensitivity,rapid read-out of results.

Another area in which specific DNA or RNA sequences are of interest ismutation and polymorphism analyses. The number of base changesdiscovered (mutations) in different genes is growing rapidly. Thesechanges are associated with genetic diseases, with diseasepredispositions and cancers, with development of drug resistance inmicroorganisms, and with genetic polymorphisms. Polymorphisms are usefulfor determining the source of a sample, e.g. in forensic analyses.Polymorphisms such as in the HLA system are essential to predict successof tissue transplants. The ability to simultaneously analyze manymutations in a gene in a simple, fast, and inexpensive way is essentialin clinical medicine and this need has stimulated the development ofdifferent methods for screening mutations, but all have seriouslimitations. What is needed are kits that are transportable andinterpretable, e.g. for use in clinics without high technologymicroscopes.

Hybridization of filter-immobilized DNA with allele-specificoligonucleotides was suggested as a way to screen for mutations. (Conneret al., 1983) However, the number of alleles that can be assayed at onetime is limited, the filters are usable only for a few times, and thereis little opportunity for complex analysis or easy interpretation ofresults.

A possible solution to large scale hybridization is to use microchipsfor DNA sequence hybridizations (SHOM, sequencing by hybridization witholigonucleotides in a microchip) (e.g. Khrapko, 1996; Yershov, 1996).The development of an array of hundreds or thousands of immobilizedoligonucleotides, the so-called “oligonucleotide chips”, permitssimultaneous analysis of many mutations (for a review, see Mirzabekov,1994). Such arrays can be manufactured by a parallel synthesis ofoligonucleotides (Southern et al., 1992; Fodor et al., 1991; Pease etal., 1994; Matson et al., 1995) or by chemical immobilization ofpresynthesized oligonucleotides (Khrapko et al., 1991; Lamture et al.,1994; Ghu et al., 1994). Glass surfaces (Southern et al., 1992; Fodor etal., 1991; Ghu et al., 1994), glass pores (Beattie et al., 1995),polypropylene sheets (Matson et al., 1995), and gel pads (Khrapko etal., 1991; Yershov et al., 1996) have been used as solid supports foroligonucleotide immobilization. However “Oligonucleotide arraytechnology has not yet lived up to its promise.” Southern, 1996 p. 115.

Some of the deficiencies in the art are unpredictability of the results,lack of knowledge of optimum conditions, and failure to demonstrateaccuracy and commercial feasibility. Moreover, analysis of the resultsof hybridization requires computer programs capable of assimilating andinterpreting multiples bits of information, and high technologymicroscopes. The microchips are neither portable, reusable, nor easilyinterpreted.

SUMMARY OF THE INVENTION

This invention embodies applications of oligonucleotide microchiptechnology wherein the microchip is a biosensor and customizedoligonucleotide microchips are designed for specific applications ofnucleic acid hybridization.

Hybridization is a process by which, under defined reaction conditions,partially or completely complementary nucleic acids are allowed to joinin an antiparallel fashion to form specific and stable hydrogen bonds.

Aspects of the invention include:

1. microchips designed so that multiple bits of genetic information areconverted to a pattern, which is interpreted as a unit, wherein theappearance of the pattern provides answers to specific questions; thisconstruction facilitates providing easily interpretable answers providedby hybridization patterns and removes some need for high technologyinstruments to interpret the results of hybridization; and

2. improved methods of hybridizing oligonucleotides in a sample tooligonucleotides on a customized microchip do not require a washing stepbut rather measure non-equilibrium melting curves (temperature curves)that do not require washing with a solution that removes immobilizedoligonucleotides from microchips; this means that microchips arereusable because the oligonucleotides, anchored within the gel elements,do not wash away, and are available for reuse. (Microchips with samplesare generally kept in solution, however, microchips can be dried andstored for many months before being reused.)

The patterns exhibited after hybridization to a microchip generally arenot directly related to the nature of the hybridizations and are notsimply converting a “yes” or a “no” signal, or a “positive” or“negative” signal to a binary outcome, nor are the patterns of thepresent invention converting a gradation of quantities to another formof gradation, e.g. calorimetric gradations. The deliberate organizationof the oligonucleotides on the microchips themselves does not transmitinformation; only after hybridization with a test nucleic acid will thehybridization signal itself form the pattern. The pattern is thendetected by a detection means which can include visual interpretationwithout the aid of additional detection instrumentation.

By choosing ordered schemes of oligonucleotide positioning on themicrochips, visual signals are simplified and enhanced, e.g. the letter“P” is observed if certain pathogenic groups are present; columns of gelelements on the chip that include the same oligonucleotide probes, willbe readily detectable as a positive linear column, if the matchingoligonucleotides are in the test sample. The visual appearance may bestrong enough to see with the naked eye, may be determined with a UPC(Universal Product Code or “bar code”) laser scanner, or with a lasergun. The wavelength of the scanner and the sensor that accepts thesignal for a bar code must be concordant with the dye or label used tohybridize the DNA.

Of course, aspects one and two do not have to be used together. Designsthat result from converting multiple amounts of genetic informationobtained by large numbers of hybridizations of oligonucleotides tosimpler, readily interpretable patterns, could be done on microchipsconstructed and analyzed by the methods used prior to the presentinvention.

Similarly, the improved methods of providing hybridization results onmicrochips could be used on microchips that are not designed to convertmultiple pieces of genetic information into a simpler pattern.

Other aspects of the invention include improved predictability,increased accuracy, and standardized factors for detection andidentification of nucleotide sequences. The improvements result fromoptimizing conditions, methods and compositions for microchiphybridization. Deliberate ordered schemes that are designed to answerspecific questions and that convert complex data to simpler patterns,are followed so that much hybridization information can be readilyobtained from a single scan of a microchip to detect hybridization ofimmobilized oligonucleotides by nucleic acids in a sample to beinvestigated. Samples include air, water, soil, blood, cells, tissue,tissue culture and a food. An aspect of the invention is that the samemicrochip can be used for hybridization for more than 20-30 times,without any noticeable deterioration of the hybridization signal becauseimmobilized oligonucleotides are not washed out or stripped. Customizedsets of microchips are obtained for specific applications. Also,parallel hybridization of nucleic acids in a sample to manyoligonucleotides on a microchip is possible, allowing replication andstandardization. For example, the sequence diversity of SSU rRNAsrecovered from different microbial populations of varying abundances isanalyzed by a single hybridization to a microchip. A large number of HLAalleles, are assayed by a single hybridization to a microchip.

The invention relates a method for identifying a nucleotide sequence ina sample using a microchip, said method comprising:

a) providing a customized matrix of oligonucleotides on the microchipdesigned to identify genetic sequences in the sample, wherein an orderedscheme positions oligonucleotides to provide a pattern to answerspecific questions after hybridization;

b) hybridizing nucleic acids extracted from the sample as such or afteramplification on said microchip; and

c) identifying the nucleotide sequences represented in said sample byanalyzing the pattern of the oligonucleotides which hybridized to thesequences, said pattern provided by signals.

The nucleic acids suitable for the practice of the invention includeDNA, mRNA, 16S rRNA sequences and other RNA species.

Customized oligonucleotide microchips are aspects of the invention. Themicrochip includes a gel-matrix affixed to a support, said matrix isformed by a plurality of gel pad element sites. The number of sites isdetermined by the number of oligonucleotides in the array. Each gelelement contains one chemically immobilized oligonucleotide of a desiredsequence, length and concentration; the gel elements being separatedfrom one another by hydrophobic glass spaces and the gel portions havinga vertical height above the plane of the interstitial spaces ofgenerally not more than 30 μm. In some applications, the same type ofoligonucleotides may be immobilized to different gel pads to form apattern.

The invention relates screening nucleic acid preparations for genes, RNAtranscripts or any other unique nucleotide sequences, for example thosethat encode microbial 16S ribosomal RNAs. Ratios of DNA/RNA or any otherunique nucleotide sequences specific for certain types of organisms aresuitable. Multiple labeling allows simultaneous detection andquantitative comparison of different nucleic acid sequences that arehybridized to a microchip.

The methods of the present invention include labeling theoligonucleotide sequence in said sample before bringing it in contactwith the array. A suitable label is a fluorescent dye. A plurality ofdifferent dyes may be used concurrently. Oligonucleotides immobilized ona customized microchip include those complementary to the beta globingene, sequences specific for Salmonella, or polymorphic HLA allelesequences.

An oligonucleotide microchip for the detection and classification ofnitrifying bacteria has a customized design wherein identifying labelsin the cells of the microchip refer to oligonucleotides selected from aclass of bacteria, and the selection is designed to answer specificquestions regarding classification.

An embodiment of an application of the present invention is detectingand identifying microorganisms in samples obtained from the environment,e.g. water, air or soil samples to check for pollutants; biologicalsamples obtained for medical diagnosis; or food samples to check forcontamination. Other applications include forensic testing to identifyDNA in samples obtained for criminal investigations, and detection ofchromosomal fragments, or single gene mutations e.g. for diagnosinggenetic diseases such as thalassemia or types of cancers. Tissue typingfor polymorphic HLA alleles for transplantation or studying humandiversity is facilitated.

The nucleic acid preparations are made from samples collected in anytype of environment, where detection and identification of themicroorganisms in that environment is of interest, or where it is likelythat new (previously unidentified) organisms may be discovered.

DNA and RNA molecules in a sample can be separated from each otherduring their isolation and labeled with different fluorescent dyes.These RNA and DNA molecules are simultaneously hybridized witholigonucleotides on a microchip that is specific to the sample to betested. The quantitative monitoring of the simultaneous hybridization ofdifferently labeled DNA and RNA with a microscope that can discriminatemulticolors at several wave lengths allows the calculation of DNA/RNAratios in the sample. For bacterial samples, this ratio determines thestate of vitality and physiological activity of the bacterium. In anembodiment, the ratio of RNA/DNA is used to discriminate the deadbacterium cells and spores from the active state of microbial growth. Inthe same way, a DNA or RNA molecule of a bacterial strain stained withone dye can be added in a calculated amount as an internal standard to asequence or sequences under investigation in which the sequences beinginvestigated stained with a different (second) dye. The fluorescencemeasurements of hybridization intensities at different wave lengths forthe standard and investigated sequences (probes) allow relativequantitative ratios to be determined.

Hybridization on microchips allows unambiguous typing of differentgroups of chosen bacteria in a sample. Microchip hybridization is asimple, fast, inexpensive and reliable method for bacterial typing.

An aspect of the invention is that there is no limitation on the numberof sequences that can be checked or the number of type:; ofmicroorganisms that can be detected. Instead of multiple sequentialhybridizations with different probes of, e.g. a 16S rRNA preparation,only one round of hybridization is; required to find out what differentsequences are in a sample. The volume of hybridizations is dramaticallyreduced and the assay requires much less RNA or DNA compared withstandard techniques. An advantage is that culturing of bacteria and geneamplification cam be avoided.

Methods of the invention significantly reduce sample preparation time,avoid the culturing of organisms collected from field situations,, andallow the identification of all species of microorganisms contained in aparticular ,,ample. Portable microchips are available for field work.

For example, oligonucleotides complementary to small subunit rRNAsequences of selected microbial groups, encompassing key genera ofnitrifying bacteria, were shown to selectively retain or hybridize withlabeled target nucleic acid derived from either DNA or RNA forms of thetarget sequences. Methods and compositions of the present inventiondiscriminate among the Genera, Nitrosomonas, Nitrobacter andNitrosovibrio sp. using fluorescently labeled nucleic acid probes thathybridize to 16S rRNA sequences. Each species has specific DNA sequenceswithin the variable region of its rRNA genes. Since the rRNAs arenaturally amplified, often present in thousand of copies per cell, theyprovide greater sensitivity, eliminating the need for amplification inmany applications.

The invention facilitates identification of organisms from environmentalsamples in a faster, and more economical approach than presentlyavailable. In addition, new species may be discovered that would behighly informative regarding taxonomic status of known as well as newlydiscovered organisms.

A diagnostic assay of the present invention for a mutation in a gene,includes the following steps:

a. designing a customized oligonucleotide microchip biosensor comprisingoligonucleotides that hybridize to a gene having the mutation, whereinthe oligonucleotides are positioned on the microchips so that patternsresult depending on what oligonucleotides are in the sample to answer aspecific question(s);

b. contacting a nucleic acid sample to the customized oligonucleotidemicrochip biosensor under conditions that allow hybridization of thenucleic acid to the microchip; and

c. determining the pattern of hybridization from which observation thepresence of specific nucleic acid sequences is inferred and the specificquestion is answered.

For diagnostic assays for genetic diseases, sequence analysis of DNA iscarried out by hybridization of PCR amplified DNA or its RNA transcriptswith oligonucleotide array microchips. Polyacrylamide gel padscontaining allele-specific immobilized oligonucleotides are fixed on aglass slide of the microchip. The RNA transcripts of PCR-amplifiedgenomic DNA are optionally fluorescently labeled by enzymatic orchemical methods and hybridized with the microchip. In the field, thechemical methods are preferred because results are obtained faster, andsome chemicals will fragment DNA at the same time which is needed forthe sample.

When melting curve experiments are performed, both matching andmismatching oligos can be immobilized in the gel pads, and both matchingand mismatching nucleic acids can be in the sample. The biochips arereusable in two types of embodiments: 1) the sample or test nucleicacids can be removed or stripped off the chip and a different testsample can be introduced and 2) the same melting point curve experimentscan be run and re-run without any washing.

When experiments are performed with a different test sample, theoriginal sample is removed from the chip by a washing or strippingprocedure using distilled water at 60° C. with an hour (or up toovernight) incubation. If the melting curve experiments are repeated (orreused) then the same sample is left in contact with the chip andappearance and disappearance of hybridization signal is observed over avariety of temperatures, usually ranging from 0°-50° C.

When the chips are incubated, in order to remove the sample nucleotides,virtually none of the immobilized oligos are removed in the process.This is because the oligos are covalently linked to the gel matrix ofthe gel pads that form the microchip.

Repeated reuse of the chips in which different samples are applied aftersequential removal is usually limited to about 50 uses, becauseeventually the amount of non-specific or background hybridization signalis greater than one-tenth of a mismatch hybridization signal. Theconditions under which a chip would not be reusable (up to 50 times) arevery few. Such conditions include allowing the chips to be cooled to−20° C. or performing experiments where the chips are heated to above70° C., conditions that have been shown to cause degradation of thechips, thus rendering them imstable.

The simultaneous measurement in real time of the hybridization andmelting curves on the entire oligonucleotide array is carried out with afluorescence microscope with a laser light source equipped with CCDcamera or a special laser scanner. Some work only with dried microchips.The monitoring of the hybridization specificity for duplexes withdifferent stabilities and AT content is enhanced by its measurement atoptimal discrimination temperatures on melting curves. Microchipdiagnostics are optimized by choosing the proper allele-specificoligonucleotides from among the set of overlapping oligomers. Theaccuracy of mutation detection can be increased by simultaneoushybridization of the microchip with at least two differently labeledsamples of normal and mutated alleles, and by parallel monitoring theirhybridization with a multi-wavelength fluorescence microscope. Theefficiency and reliability of the sequence analysis was demonstrated bydiagnosing β-thalassemia mutations and HLA polymorphisms. Determininglevels of gene expression is an aspect of the invention.

Because the methods of the present invention require only a simpleprocedure of hybridization and because only one round of hybridizationis necessary, it is fast and inexpensive. Because the invention allows alot of information to be obtained from one experiment, in a simplepattern as compared to the analysis of hundreds of data points, it hasincreased efficiency. The invention is reliable because the microchipsare reusable. Immobilized oligonucleotides are not washed out. There isno waste of hybridization probes, therefore the microchip hybridizationis inexpensive and non-isotopic detection simplifies all procedures.

Effective and precise sequence analysis by the hybridization of a probewith rather short microchip-immobilized oligonucleotides depends on manyfactors. Major factors are the reliability of the discrimination ofperfect duplexes from duplexes containing mismatches, differences instability of AT- and GC-rich duplexes, the efficiency of thehybridization, and simplicity in the preparation of the labeled samplesfor hybridization.

Identification of base variations is significantly improved by parallelmeasuring of the melting curves of the duplexes formed on the entireoligonucleotide array, as well as by monitoring the simultaneoushybridization of two differently labeled samples at two wavelengths andby choosing proper allele-specific oligonucleotides.

Other factors to be considered for operation of the invention include(1) regulating the flow of the fluid containing a sample to be testedover the microchip during the hybridization; and (2) control of thetemperature of the microchip gel layer and the fluid layer, in adifferential manner, by placing a cooling and heating apparatus adjacentto the gel layer and the top fluid layer. The gel layer temperature iscontrolled in a uniform or gradient manner by a heating/cooling deviceattached to the glass plate substrate of the gels. For field work, theoptimum temperature for a particular question is determined previouslyin a laboratory.

A definition of “customized microchip” is a microchip of gel elements ona support, wherein the oligonucleotides are immobilized in gel elementsaccording to an ordered scheme such that multiple bits of informationare ordered to a simpler pattern to answer a specific question.

Removal of test or sample nucleic acids from microchip is accomplishedby an incubation step carried out using distilled water for at least onehour (up to overnight) at 60° C. (This procedure is analogous to thestep of “stripping” a filter for re-use in the standard technique ofprobing a Southern blot.) The immobilized oligonucleotides in the gelmatrix are not removed by this incubation as the oligonucleotides arecovalently linked to the gel substrate.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A & 1B show non-equilibrium melting curves of duplexes of RNAwith microchip oligonucleotides.

FIG. 2 shows an example of four melting curves for 75-nt-long RNAfragments hybridized with the microchip oligonucleotides. The RNA wasderived from a patient having the IVS I/2 T/A mutation in the β-globingene. The curves were normalized to the initial hybridization signals.Melting curves 1 and 3 correspond to perfect duplexes; curves 2 and 4correspond to duplexes containing internal T-T or G-T mismatches,respectively. The curves for the perfect and mismatched duplexes areshifted by about 10° C. from each other.

FIGS. 3A-3D show hybridization of fluorescein labelled 16S rRNAs to amicrochip. The microchip with immobilized probes (see Table 1 and Table2) was hybridized sequentially to in vitro transcribed 16S rBNA ofNitrosovibrio tenuis (A), Nitrosomonas europaea (B), E. coli (C), andwith E. coli rRNA recovered from isolated ribosomes (D). The panels tothe right display the member of mismatches between each probe and theRNA.

FIGS. 4A and 4B show hybridization of the mixture of differentlylabelled E. coli and Nitrosovibrio tenuis rRNAs to the microchip at 10°C. and 40° C., measured simultaneously by multicolor detection. A. Themicrochip was hybridized with a mixture of fluorescein labelledNitrosovibrio tenuis and tetramethylrhodamine labelled E. coli 16S rRNAand washed serially at the indicated temperatures, arbitrary units offluorescence intensities. B. The ratio of the hybridization intensitiesof Nitrosovibrio tenuis (I_(Nt)) to E. coli (IE. coli) 16S RNA measuredat 10° C. and 40° C. R=(I_(Nt)/l_(I) E. coli).

FIG. 5 illustrates the concentration effect of the immobilizedoligonucleotides on the hybridization intensities. A microchip withdifferent concentrations of immobilized oligonucleotides was hybridizedwith N. tenuis 16S rRNA labelled with fluorescein and washed at 20° C.Curve 1 corresponded to Nsv443 (nitrosovibrio-like) probe, curve2-Bac338 (Bacteria), curve 3-Nso1225 (ammonia oxidizers), curve4-Uni1390 (all life), and curve 5-Nsm-156 (nitrosomonas), a.u.—arbitraryunits of fluorescence intensities.

FIG. 6 shows the sequences of β-globin alleles specifyingoligonucleotides that were immobilized on a microchip.

FIG. 7 shows the experimental design to detect β-globin mutations usingoligonucleotide microchips.

FIG. 8 shows results of gene expression studies.

FIG. 9 shows 18 short HLA oligonucleotides.

FIG. 10 shows HLA oligonucleotides hybridized to the microchips.

FIG. 11 illustrates a closed microchamber 1 containing a microchip witha gel array 3 on a glass support 4; ports 2 are used merely to providewetting solution.

FIG. 12 shows an ordered scheme in which a letter “P” will be detectedif there is a group of hybridizations of oligonucleotides from a samplethat are oligonucleotides from pathogens.

FIG. 13 illustrates an ordered scheme on a microchip wherein thepresence of B. anthracis.

DESCRIPTION OF THE PREFERRED EMBODIMENT

This invention relates to using customized oligonucleotide microchips asbiosensors for the detection and identification of nucleic acidsspecific for different genes, organisms and individuals in theenvironment, in food and in biological samples. “Environment” includeswater, air and soil. Biological samples include blood, skin, tumorsamniotic fluid, tissues, cells and cell cultures. Detection of sequencesin nucleic acids is used to identify microorganisms in a sample, todiagnose genetic defects or polymorphisms, to detect gene expression andfor forensic studies.

Means for detecting a pattern generated by signals from hybridizationwithin individual gel elements in a microchip of the present inventioninclude a laser scanner, e.g. a laser “gun” such as used to scan barcodes, a CCD camera coupled to a fluorescent microscope. In the field,the naked eye or a scanner is used.

The invention relates to a deliberate and informative arrangement ofoligonucleotides immobilized on a microchip, such that uponhybridization with oligonucleotides in a test sample, a pattern isproduced that can be interpreted with a suitable means. Hybridizationmay be detected by letter (FIG. 12), design or bar code pattern (FIG.13) wherein columns “1” and “3” are dark bars signifying the presence ofa pathogen, and the specific pathogen in “1” is a different anthraxspecies from that in “3.” By immobilizing all of one type ofoligonucleotide in a column, for example, the pattern is readilydetected as a linear column, as contrasted to detecting hybridization ina single small gel element or elements, which requires a microscope todetect it, and computer programs to analyze it.

A nucleic acid hybridization is a highly specific and sensitiveprocedure and allows a specific sequence to be detected and identifiedamong other millions of sequences in a genome of an organism. However,nucleic acid hybridization is a useful but quite a cumbersome procedure.This drawback can be overcome by using oligonucleotide microchips asbiosensors for different microorganisms. Within a small area of a fewsquare millimeters or centimeters, hundreds and thousands of syntheticoligonucleotide probes are immobilized that are specific to ribosomalDNA or to other specific nucleic acids. Subsequent hybridization of aDNA or RNA molecule to the microchip enables a menu of oligonucleotidesto be identified in a sample. Instead of having to interpret hundreds orthousands of individual hybridizations, a relative simple patternproduced by hybridizations is analyzed.

For bacterial assays, pure culture microorganisms, purified targetnucleic acid or even synthetic oligonucleotides are useful as internalstandards, serving to estimate the efficiency of nucleic acid isolationor the absolute amount of target nucleic acid recovered.

The customized oligonucleotide microchips are produced by chemicalimmobilization of presynthesized oligonucleotides, or by directsynthesis of oligonucleotides on a microchip. If a microchip containsrather long oligonucleotides, the former methods are the methods ofchoice because before immobilization, the oligonucleotides are purifiedand checked for their quality.

Methods and technologies have been developed for microchipmanufacturing, hybridization of fluorescently labeled DNA and RNA withthe microchips and monitoring the hybridization with a fluorescencemicroscope equipped with CCD-camera, computer and proper software (seeU.S. Pat. No. 5,552,270 her,.in incorporated by reference).

The oligonucleotide microchips consist of many polyacrylamide gel padelements generally of the size of 40×40×20 μm and larger. The elementsare chemically fixed on a glass surface. Each microchip gel elementcontains a specific presynthesized oligonucleotide that is immobilizedthrough a covalent bond. Hundreds of microchips containing hundreds andthousands of different immobilized oligonucleotides can be manufacturedby a specially devised robot. The gel array also offers severaladvantages over formats using an in situ synthesis of theoligonucleotide array. The synthetic oligonucleotides are purified bygel electrophoresis or HPLC prior to immobilization on the microchip.This provides for stringent quality control of oligonucleotide purityand insures high specificity. The polyacrylamide gel support has acapacity of immobilized oligonucleotides from 0.03 pmol up to 10 pmolper 100×100×20 μm gel pad. This offers improved quantification andbetter discrimination between perfect and mismatched duplexes. It alsoprovides a way to normalize differences in hybridization signalintensities.

Oligonucleotide microchip technology for sequencing by hybridization isavailable to identify the presence of microorganisms in a sample of anytype, or to find new species. As shown in the examples herein, thehybridization of DNA or RNA extracted from even a very complicatedmixture to a specific oligonucleotide probe has resulted in unambiguousidentification of microorganisms. The nucleotide sequence of themicroorganisms for genes encoding a small subunit of ribosomal 16S rRNAforms the basis for a microchip biosensor. Instead of direct sequencingof the gene, hybridization analysis of DNA or RNA samples witholigonucleotides specific for the microorganisms is performed. This newtechnology provides efficient microbial analysis and environmentalmonitoring. Fluorescently labeled DNA and RNA samples frommicroorganisms are hybridized with microchips containingoligonucleotides specific for several microorganisms. Thesemicroorganisms are reliably identified by microchip hybridizationpatterns. Microorganism biosensor technology is developed, reusablecustomized microbial oligonucleotide microchips are produced by methodsof the present invention, and methods are developed for simultaneousquantitative and qualitative microchip analysis of hundreds andthousands of microorganisms in a sample and for discovery of new ones.

EXAMPLES

The following examples are presented as illustrations of aspects of theinvention, rather than limitations of the invention. Other applicationsinclude detection of genetic mutations such as are characteristic ofhemoglobin disorders; detection of genetic polymorphisms such as HLA;investigation of gene expression; detection of causative agents ofdiseases; forensic studies; and detection of microbial pollutants.

Example 1

Preparation of an Oligonucleotide Microchip Biosensor

Oligonucleotides are synthesized using a 394 DNA/RNA synthesizer(Applied Biosystems). The synthesis of oligonucleotides forimmobilization began with 3-methyluridine at the 3′-terminal position.

In one embodiment, fluorescently labeled RNA was prepared using T7 RNApolymerase. Template DNA (133 and 75 bp long) for in vitro transcriptionwas prepared by PCR amplification with the nested primers T7-V2L-45,5′-GGAATTCCTAATACGACTCACTATAGGGACACCATGGTGCACCTGACTCC-3′ (SEQ ID NO: 5),as well as with the common reverse primer T7-V2L-1035′-GGAATTCCTAATACGACTCACTATAGGGAGGTGAACGTGGATGAAGTTGG-3′ (SEQ ID NO: 16)AND 5′-TCTCCTTAAACCTGTCTTGTAACC-3′ (SEQ ID NO:17). Templates werepurified using a QIAquick PCR purification kit (QIAGENE) according tothe manufacturer's protocol. The RNA polymerase reaction was performedusing the MEGAshortscript™ T7 kit (Ambion) with fluorescein 12-UTP(Molecular probes). Fluorescently labeled ssDNA (single stranded DNA)fragments were prepared by single primer reamplification.

A polyacrylamide gel micromatrix was prepared by photopolymerization ofa solution of 4% acrylamide (acrylamide/bisacrylamide 19/1), 40%glycerol, 0.0002% methylene blue, and 0.012% TEMED in 0.1 Msodium-phosphate buffer, pH 7.0. The mixture was applied to an assembledpolymerization chamber illuminated with U.V. light.

Two types of microchip matrices (micromatrices) were routinely preparedwith gel pad elements of about 60×60×20 μm and 100×100×20 μm that werespaced by 120 and 220 μm, respectively. About 1 nl of activatedoligonucleotide solution was transferred to a gel element using either arobot or a simple manual device.

The device includes a Peltier thermostated pin placed under a binocularlens in conjunction with a micromanipulated holder, a power supply, anda refrigerated circulator.

The manufacture of microchips of gel-immobilized oligonucleotidesbasically consists of three steps; shaping the desired topology ofoligo-nucleotides on a gel micromatrix; loading microvolumes ofoligonucleotide solutions onto the micromatrix, and immobilizing withinthe gel oligonucleotides containing the active 3′ or 5′ terminalaldehyde or amine groups.

To avoid the exchange of different oligonucleotide solutions applied onadjacent gel pads, the pads are separated on the micromatrix by ahydrophobic glass surface. Two-dimensional scribing or laser evaporationis used for micromatrix preparation, but these procedures require rathercomplex equipment and experienced personnel. The photopolymerizationmethod significantly simplifies the procedure and makes it accessible toa biochemical laboratory.

Microfabrication by mask-directed photopolymerization (e.g., aphotoresist method in microelectronics) is a well developed technique.From several acrylamide photopolymerization techniques tested,modified—methylene—blue induced photo-polymerization produced the bestresults for micromatrix manufacture. The gel matrix consists of gel padsphotopolymerized on a glass slide. The gel pads are formed according tothe mask topology due to the lack of photopolymerization in placescovered by a nontransparent grid.

The microchip is manufactured by applying the activated oligonucleotidesolutions onto the micromatrix of gel elements containing activehydrazide or aldehyde groups. A simple device exists for manual loadingof up to 100 different oligonucleotides on a micromatrix. The transferis carried out by the hydrophilic upper surface of a pin that is firstimmersed into, and is wetted with, an oligonucleotide solution, and thenis withdrawn from the solution and brought into contact with the gelsurface. This transfers about 1 nl of oligonucleotide solution with areproducibility of ±10%. The temperature of the pin is maintained nearthe dew point of the ambient air to avoid the evaporation of thismicrovolume solution in the course of transfer.

The oligonucleotides are positioned according to a design whereinhybridization pattern data will be reduced to a readily interpretablepattern.

Example 2

The Hybridization of Microchips with DNA and RNA using a HybridizationBuffer

Fluorescently labeled DNA or RNA (5 μl, 0.1-1 pmol/μl were hybridized toa microchip at +5° C. in a hybridization buffer containing 1 M NaCl, 1mM EDTA, 1% Tween-20, and 10 mM sodium phosphate at a pH of 7.0, forbetween about 2-24 h. The microchip was covered with a cover glass or aTeflon sheet so that a 300-μm space is above. Then the hybridizationsolution containing DNA or RNA fragments was substituted with 10 μl ofcooled hybridization buffer. The microchip with the cover glass wasplaced on a thermostabilized table. Hybridization was monitoredquantitatively using a specially constructed multicolor epifluorescentmicroscope with a 4×4 mm observation field equipped with a CCD cameraand suitable software.

Example 3

Analysis of Melting Curves; a Hybridization Buffer is Not Required

The polyacrylamide gel used on a microchip provides more than 100 timeshigher capacity for three-dimensional immobilization of oligonucleotidesthan does a two-dimensional glass surface. The high concentration ofimmobilized oligonucleotides facilitates the discrimination ofmismatched duplexes and enhances the sensitivity of measurements on themicrochips. This allows the use of a CCD-camera-equipped fluorescencemicroscope (Yershov et al., 1996) although it is less sensitive thanlaser scanning systems (Lipshutz et al., 1995), but offers the advantageof monitoring the hybridization on a microchip at different temperaturesin real time for measurement of the melting curves. Melting curves aredefined herein as produced by plotting the amount of duplexes[fluorescent intensity] versus temperature. The procedure, the software,and the hybridization microchamber (Yershov et al., 1996) have all beendeveloped for recording melting curves at a wide range of temperaturessimultaneously for perfect and mismatched duplexes formed uponhybridization of a probe with all microchip oligonucleotides.

A significant amount of time is needed for the microchips hybridizedwith rather long RNA or DNA probes to achieve equilibrium. Therefore,non-equilibrium dissociation melting curves were measured. However, theyare riot far away from equilibrium where some difference in heating ratedid not significantly affect the results. The melting curves forhybridization of, for example, synthetic 19-mers with the microchipoligonucleotides reached equilibrium under the same conditions that wereused for measuring non-equilibrium RNA and DNA melting curves. Themelting curves can also be measured after a few minutes, far away fromequilibrium, if an internal standard is added to a tested sample. Thisstandard can be a differently labeled RNA of a normal allele. Thissignificantly speeds up the identification of nucleic acid base changes.

Example 4

Choice of Optimum Melting Temperatures for Non-Equilibrium Hybridization

This invention embodies an improvement in the SHOM technology in whichhybridizations between an array of gel-immobilized nucleotides (amicrochip) and the unknown nucleotides to be tested are measured atoptimal, discriminatory melting temperatures. This improvement isachieved by parallel measuring of the melting curves of the duplexesformed by hybridization on the entire oligonucleotide array, as well asby monitoring the simultaneous hybridization of two samples ofnucleotides labeled with different fluorochromes, and judicious choiceof proper allele-specific oligonucleotides as the immobilized probes.The fluorochromes chosen for the labeling emit light of sufficientlydiffering wavelengths, that both types of labels can be measured in thesame reaction mixture.

The greatest discrimination between perfect and mismatched duplexes wasachieved at a temperature at which the intensity of the hybridizationsignal from a perfect duplex dropped to one-tenth of its initial value;at such a temperature, the hybridization intensities from mismatchedduplexes usually approached the background level. The temperature atwhich the initial signal of hybridization drops by a factor of 10 istermed the discrimination temperature (Td.).

In the case of beta-thalassemia mutation detections described in Example6 herein: (1) RNA transcripts of PCR-amplified DNA were hybridized withimmobilized oligonucleotides; (2) the Td values for perfect 40% and 70%GC-rich duplexes were 52° and 64°, respectively; (curves 1 and 3 inFIGS. 1A and 1B); (3) the immobilized oligonucleotides were chosen fromamong a set of overlapping sequences; and (4) the two samples includedin the reaction mixture were a mutated allele RNA labeled with onefluorochrome and a sample of the normal allele RNA labeled with adifferent fluorochrome.

The Td is determined by hybridization with an RNA sample if an allelicDNA is available. If such DNA is unavailable, the Td can be measuredfrom the hybridization data resulting from experiments performed withsynthetic oligonucleotides corresponding to the mutated allele ofinterest.

The dissociation curves for perfect and mismatched duplexes are parallelat the range of about 10° (in the middle of the curves) when plotted ona semilogarithmic scale. At this 10° C. range, the ratios of the signalsfor perfect and mismatched duplexes remain rather constant. This makesthe discrimination procedure robust to some inaccuracies in determiningTd. The discrimination temperature depends on experimental conditions(rate of heating, ionic strength, probe concentration, extent offragmentation, and so forth) which can vary from one experiment toanother. However, these variations affect Td and the relativeintensities of the hybridization signals to a similar extend for allmicrochip elements and therefore do not significantly distort thediscriminations. Therefore, to provide a reference Td, theoligonucleotides CD26(N) and CD26 G/A, which form perfect and mismatchedduplexes, respectively, with all RNAs tested, were introduced into themicrochip.

Since Td is robust to some inaccuracy in measurements, 19-meroligodeoxynucleotides were used in these experiments instead of moreexpensive 19-mer oligoribonucleotides. There are differences in thestability of DNA-DNA homoduplexes relative to DNA-RNA heteroduplexes(Lesnik and Freier, 1995). The pattern of hybridization of the microchipwith RNA derived from patients and with 19-mers was rather similar tothat from the 10-mers. Hybridization with corresponding syntheticoligonucleotides is preferred as a control when a mutation is identifiedin an RNA sample by its hybridization with a diagnostic microchip.

A mixture of fluorescently labeled RNA samples was prepared from twopatients; the first sample was TMP-labeled RNA from a patient that ishomozygous for the normal CD26 area of the beta-globin allele; thesecond sample is fluorescein-labeled RNA from a patient that isheterozygous for the normal CD26 area and a mutation CD26 G/A alleles.This mixture was hybridized with a microchip consisting of two microchipelements that contained the following immobilized oligonucleotides:SEQUENCE A sample 5′-GGCCTCACCA-3MeU-3′ (SEQ ID NO: 1) (element)-CD26(N-normal) B sample 5′-GGCCTTACCA-3MeU-3′ (SEQ ID NO: 2) (element)-CD26(G/A-mutant)

Usage of different filters during the registration of the signal,allowed the independent, simultaneous registration of the sample, whichwas marked with the different dyes; TMP (red) and fluorescein (green),on the same element of the microchip. FIG. 2 demonstrates 10 theinteraction of sample 1, with the A microchip element; Graph 2demonstrates the interaction of sample 2 with the A microchip element;Graph 3 demonstrates the interaction of sample 2 with the B microchipelement; and Graph 4 demonstrates the interaction of sample 1interaction with the B microchip element.

Example 5

Use of a Customized Microchip Matrix Biosensor to Identify NitrifyingMicroorganisms

The results in this example were obtained using methods previouslyavailable, not the non-equilibrium melting curves. Microorganisms thatdegrade nitroaromatic compounds include Pseudonomas, Arthrobacter,Nocarida, Myco-bacterium, and fungi.

Previously, methods for detection of these bacteria were tedious andinaccurate. For example, to detect Pseudonomas capable of degradingnitroaromatic compounds, 2-nitroluene was tested as a sole carbon,energy and nitrogen source. It was difficult to isolate the bacteriafrom soil samples to perform the test.

Nitrifying bacteria have proved particularly difficult to study usingcultivation techniques, such as most probable number (MPN) and selectiveplating because of their long generation times and poor countingefficiencies,. Thus, a rapid and non-culture dependent enumerationtechnique for nitrifiers could greatly facilitate research in theirecology.

Microchips with 100×100×20 μm gel pads (alternatively 60×60×20 μm),fixed on a glass surface and containing a set of 10 oligonucleotides15-20 bases-long were manufactured for bacterial typing experiments. Theset included oligonucleotides complementary to different regions of 16Sribosomal RNA. Since rRNA's are naturally amplified, and often arepresent in thousands of copies per cell, they provide great sensitivityand eliminate the need for amplification in many applications. Oneoligonucleotide is represented in most living organisms, another istypical for most of bacteria and the rest belong to nitrosos(nitrifying) bacteria only. The group of nitrosos bacteriaoligonucleotides consists of two oligonucleotides typical ofnitrobacter, two typical of nitrosomonas and one typical ofnitrosovibrio. One oligonucleotide was complementary to an antisensestrand of rDNA for hybridization with ribosomal dsDNA, that was PCRamplified from genomic or cDNA.

The following scheme for an ordered oligonucleotide loading (placing ona chip) is useful for bacterial (or organism, species) typing. In themicromatrix design shown in Table 1, the first oligonucleotidescharacterize the highest order (i.e. to distinguish a living organism).[Uni 1390-CIII]. Reducing the order step by step down to the lowestlevel, i.e. from family, to genus, to species provides furtherdiscrimination of oligonucleotides that are present in a sample beinginvestigated. For example, for oligonucleotides used to classifynitrifying bacteria, a bacterial oligonucleotide would be in the nextposition. [Bac 338-CI and NonBac338-CII]. Oligonucleotides specific tonitrobacter [Nb1000—AI and NIT3-AII] and ammonia oxidizers [NEU23-AIII,Nso1990-AIV and Nso1225-BI] follow in any order. Finally,oligonucleotides specific to Nitrosomonas [Nsm156-BII] and Nitrosovibrio[Nsv443-BIII] complete the micromatrix design.

The microchip was evaluated using three different rRNA preparations(phenol extracts of cellular RNA, RNA isolated from purified ribosomes,and in vitro transcripts of cloned ribosomal DNA), and both fragmenteddouble-stranded and single-stranded DNA. Hybridizations were performedin a formamide buffer at low temperature in order to enhance microchipdurability and decrease RNA degradation. Although all DNA and RNApreparations could be used, the best discrimination was observed for invitro transcribed rRNAs using the hybridization conditions evaluated inthis study.

The hybridization of the microbial microchips was carried out with fivedifferent preparations of target nucleic acids. Ribosomal RNA and totalRNA were recovered from cells. RNA transcribed in vitro as well assingle- and double-stranded PCR-amplified 16S rDNA were obtained fromplasmids containing the cloned 16S rRNA gene. All of these sample typesprovided a comparatively reliable identification of the microorganismsby their hybridization with the microchip-immobilized oligonucleotidesand could be used for different purposes. For example, the rRNA providesa naturally amplified target. Also, since cellular ribosome content iswell known to vary with growth rate, it is generally thought that directquantification of rRNA serves to identify the more active environmentalpopulations. In contrast, analysis of PCR amplified rDNA provides a moregeneral measure of all microorganisms present in a sample.Alternatively, these measures could be combined. For example, the RNAand DNA components of an environmental sample could be isolated andlabelled with different fluorescent dyes. Following their combinedhybridization, the resulting ratio of RNA and DNA hybridizing to anindividual gel element could be used to infer the physiological statusof the corresponding microbial population.

Table 2 shows the sequences of the oligonucleotides and othercharacteristics of them. TABLE 1 MICROMATRIX DESIGN FOR NITRIFYINGMICROORGANISMS I II III IV A Nb1000 NIT3 Nso190 B Nso1225 Nsm156 Nsv443C Bac338 NonBac338 Uni1390

TABLE 2 Oligo- nucleotide Microchip Name and location Td¹ PositionSequence (5′ to 3′) Specificity Table 1 C. Nb1000 5′-tgc gac cgg tcatgg-3′ Nitrobacter A-I 42° (SEQ ID NO: 6) NIT3 5′ cct gtg ctc cat gctccg-3′ Nitrobacter A-II 66°² (SEQ ID NO: 7) NEU23 5′-ccc ctc tgc tgc actcta-3′ Ammonia oxidizers A-III 66°² (SEQ ID NO: 8) NS0190 5′-cga tcc cctgct ttt ctc-3′ Ammonia oxidizers A-IV 62° (SEQ ID NO: 9) NSO1225 5′-cgcgat tgt att acg tgt ga- Ammonia oxidizers B-I 51° 3′ (SEQ ID NO: 10)NSMO156 5′-tat tag cac atc ttt cga t-3′? Nitrosomonas B-II 46° (SEQ IDNO: 11) NSV443 5′-ccg tga ccg ttt cgt tcc-3′ Nitro-sospira-like B-III52° (SEQ ID NO: 12) BAC338 5′-gct gcc tcc cgt agg gat-3′ Bacteria C-I54° (SEQ ID NO: 13) NonBAC3 5-′act cct acg gga ggc agc-3′ Eub338 C-II54° 38 (SEQ ID NO: 14) complementary strand UNI1390 5′gac ggg cgg tgtgta caa-3′ all life (with a C-III 44° (SEQ ID NO: 15) few exceptions)¹Experimentally determined.²Estimated from in situ hybridization.

A number of hybridization conditions were tested in terms of efficiencyand specificity of hybridization. Hybridization in formamide containingbuffer at low temperature gave good results. Hybridizations wereperformed at 5° centigrade in 33% formamide. RNA samples and covalentbonding of oligonucleotides with the support (hence durability ofmicrochips) are more stable at low temperatures. In addition, theseconditions were favorable from a point of view of RNA stability andmicrochip durability similar to other RNA molecules at low temperaturesof about 0°-5° C.

The hybridization on a microbial microchip was carried out with in vitroRNA transcripts of 16S rDNA of different nitroso bacteria, total RNAextracts and ribosomal RNA extracted form E. coli and Desulfovibriavulgaris as well as PCR amplified double or single stranded DNA of 16SrDNA.

The probes for ammonia oxidizing bacteria show different discriminationspecificity under different conditions. FIG. 3 shows the fluorescence ofindividual gel elements on the microchip following hybridization to the16S rRNAs of Nitrosovibrio tenuis (A), Nitrosomonas europaea (B), and E.coli, either in vitro transcribed (C) or recovered from isolatedribosomes (D). The same microchip was used for each hybridizationfollowing washing with distilled water. Each microchip was routinelyused for up to 20-30 hybridization experiments. The appropriate patternof hybridization was observed for all gel elements shown, despite asignificant difference in dissociation temperatures (Tds) previouslydetermined using membrane support hybridization (Table 1). For hybridsof comparable stability, discrimination is generally achieved by washingat increasing temperatures (described below) or by simultaneouslyevaluating their melting characteristics, since the fluorescenceanalyzer can monitor hybridization signals in real time.

FIGS. 4A and 4B shows the results of an experiment evaluating the effectof increasing washing temperature on target RNA retention. A mixture ofNitrosovibrio tenuis and E. coli 16S rRNA labelled with differentfluorescent dyes (fluorescein and tetramethylrhodamine, respectively)was hybridized to the chip at 5° C. The hybridization solution was thenreplaced with washing buffer and the retention of each RNA species wasmeasured following each 10° C. incremental increase in temperature (upto 60° C.) using multicolor detection. Nonspecific hybridization of E.coli rRNA to Nso1225 (ammonia oxidizer), Nsm156 (nitrosomonas), andNonBac338 (anti-sense) was observed following the 10° C. wash. However,this nonspecific hybridization was significantly reduced following the40° C. wash. In like manner, the 16S rRNA of Nitrosovibrio tenuishybridized to Nitrosomonas (Nsm156) at 10° C., but was reduced to nearbackground (compared to NonBac338) following the 40° C. wash. Using themethods of the present invention, hybridization buffer is not required.A more complete correction for differences in stabilities of duplexescan be carried out by measuring the equilibrium or non-equilibriummelting curves for all microchip elements. This would provide a basis tocompensate for the various factors influencing individual duplexstability, e.g., their length, GC-content, and competition withsecondary and tertiary structures in RNA and DNA.

FIG. 4B shows the ratios of hybridization intensities of fluoresceinlabelled Nitrosovibrio tenuis to tetramethylrhodamine labelled E. coli.with different microchip oligonucleotides at 10° C. and 40° C. (theratios are derived from the data presented on FIG. 4A. These ratios werenot changed significantly for oligo-nucleotides specific to bacteria andall living organisms between 10° C. and for more stringent conditions at40° C. However, the ratio is dramatically increased at 40° C. (comparedto 10° C.) for oligonucleotides specific to ammonia oxidizers andnitrosovibrio. This increase reflects the greater duplex stability ofNitrosovibrio tenuis RNA with the complementary oligonucleotidescompared with E. coli. RNA. Although the nitrosomonas ratio increases,the signal originating from each labelled RNA is near background. Thisexperiment demonstrates that the inclusion of second dye-labelled RNA,either isolated from cells or synthesized, could be used as an internalstandard for quantitative assessments of hybridization patterns.

Variable hybridization to the different gel elements is the expectedconsequence of using a single hybridization condition to evaluate anarray of probes, each having different kinetics of association anddissociation. To some extent these difference can be normalized byvarying the concentration of oligonucleotides in the individual gelelements. For example, the relatively low hybridization signals ofNso1225(b-I) and Uni1390 (c-III) compared to Nsv443 (b-III) could eachbe elevated by increasing the amount of the correspondingoligonucleotides probes immobilized in the gel. This approach wasevaluated by synthesizing a microchip with selected probes immobilizedat several different concentrations, up to 6 times higher than that usedin the experiments previously described. This was accomplished bymultiple applications of the standard loading solution (100 pmol/μlprobe) to each gel element. Comparable hybridization of Nso1225 (ammoniaoxidizer) and Nsv443 (nitrosovibrio-like) was achieved following threeapplications of the Nso1225 probe (FIG. 5).

Similarly, two applications of Bac338 (bacteria) and five of Uni1390(all life) resulted in hybridization comparable to Nsv443.

Strains used. Escherichia coli, Desuifovibrio vulgaris strain PT2,Nitrosovibrio tenuis strain NV 12, Nitrosomonas europaea strain ATCC19718, and Nitrosomonas strain C-56 were used as sources of nucleic acidfor these experiments.

RNA preparation. Total cellular RNA was isolated by phenol/chloroformextraction. For some of the samples, a ribosome enrichment was performedbefore RNA extraction. Forty ml of log phase growth E. coli or D.vulgaris strain PT2 was centrifuged at 3500 g for 10 min. andresuspended in 4 ml of 40 C ribosome buffer. Ribosome enrichment bufferconsisted of 20 mM MgCl2, 50 mM KCl, 50 mM Tris at pH 7.5, and 5 mMβ-mercaptoethanol in diethyl pyrocarbonate treated double-distilledwater. The cell suspension was divided between 4 screwtop microfugetubes and 0.5 g of 0.1 mm ZrO2 beads were added. The cell suspensionswere disrupted for 2 min., put on ice for 5 min., and disrupted againfor 2 min. The cell suspensions were centrifuged at 14000 g for 10minutes. The supernatant, which contained the ribosomes, was recoveredand transferred to ultracentrifuge tubes. Ribosomes were pelleted byultracentrifugation in ribosome buffer at 55,000 rpm (201,000 g average)for 50 minutes in a Beckman Optima Series TL swinging bucket rotor(Beckman, Fullerton, Calif.), for a Svedberg sedimentation factor of70S. After centrifugation, the supernatant was discarded and the RNA wasrecovered from the pelleted ribosomes by extraction with pH 5.1phenol/chloroform. Quality and quantity of extracted RNA was evaluatedby polyacrylamide gel electrophoresis and ethidium bromide staining.

Cloning of 16S rDNA and in vitro production of RNA transcripts. DNA wasextracted from E. coli, Desulfovibrio vulgaris PT2, Nitrosovibrio tenuisNV12, Nitrosomonas europaea 19718, and Nitrosomonas strain C-56 cellpastes using a guanidine/diatom method. Near-complete 16S rDNA genes(ca. 1500 base pairs) were recovered from each by PCR amplificationusing S-D-Bact-0011-a-S-17 (GTTTGATCCTGGCTCAG)(SEQ ID NO:3) andS-D-Bact-1492-a-A-21 (ACGGYTACCTTGTTACGACTT)(SEQ ID NO:4′) as primersand a premixed PCR amplification buffer (Pharmacia Biotech Inc.Piscataway, N.J.), consisting of 0.2 mM Mg++, 2.5 mM each dATP, dCTP,dGTP, dTTP, 0.2 mM of each amplification primer, and 2.5 units of TaqDNA polymerase (Pharmacia). Temperature cycling was done in an IdahoTechnology thermocycler (Idaho Falls, Id.) using 30 cycles of 15 sec at94° C., 20 sec at 50° C., 30 sec at 72° C. The PCR-products were clonedin a pCR plasmid (Invitrogen, San Diego, Calif.) according tomanufacturers instructions. Plasmids were isolated using the Wizard kit(Promega, Madison, Wis.) and used for in vitro transcription of thecloned SSU rRNA genes.

DNA oligonucleotide probes. All probes were complementary to the SSUrRNAs and previously characterized using a membrane hybridizationformat. Fives probes hybridize to different groups of ammoniaoxidizingbacteria within the beta-subdivision of the Proteobacteria.S-G-Nso-190-b-A-19 (Nso190) ,td S-G-Nso-1225-a-A-20 (Nso1225) encompassall sequenced ammoniaoxidizers of the beta-subclass of Proteobacteria,probe S-G-Nsm-156-a-A-19 (Nsm156) identifies members of the genusNitrosomonas (also including Nitrosococcus mobilis), probeS-G-Nsv-443-a-A-20 (Nsv443) is specific for theNitrosovibriol/Nitrosolobus/Nitrosospira group, and probeS-G-Nsm-653-a-A-18 (NEU23) is specific for the halotolerant members ofNitrosomonas. Probes for members of genus Nitrobacter (nitriteoxidizing) were S-G-Nit-1000-b-A-15 (Nb1000) and S-G-Nit-1035-a-A-18(NIT3). Other probes used were S-D-Bact-0338-a-A-18 (Bac338) whichhybridizes to members of the bacterial domain; S-D-NBac-0338-a-S-1 8(NonBac338), complementary to the antisense strand of the Bac338, andS-*-Univ-1390-a-A-18 (Uni1390) complementary to the SSU RNA of nearlyall characterized living organisms, with the exception of some protists.

RNA and DNA labeling and fragmentation. Single stranded DNA was preparedby asymmetric PCR according to Ausubel et al. (1994) using a 100 timesexcess of the forward primer. Briefly, DNA was partially depurinated in80% formic acid for 30 min. at 20° C., then incubated in 0.5 Methylenediamine hydrochloride (pH 7.4) for 3 hr at 37° C., followed by30 min. at 37° C. in the presence of 0.1 M NaBH4. Fluoresceinisothiocyanate was incorporated into fragmented DNA by incubation inabsolute DMSO at room temperature for 1 hr.

RNA was fragmented by base hydrolysis and dephosphorylated with bovinephosphatase. Fragmented RNA was oxidized by NaIO4 and labeled either byethylenediamine mediated coupling of 6-carboxyfluorescein (FAM)succinamide or by direct incorporation of tetramethylrhodamine-hydrazide(TMR).

Microchip fabrication. A matrix of glass-immobilized gel elementsmeasuring 60×60×20 or 100×100×20 μm each and spaced apart by 120 or 200μm respectively was prepared. The polyacrylamide gel was activated bysubstitution of some amide groups with hydrazide groups byhydrazine-hydrate treatment. Oligonucleotides were activated byoxidizing 3′-terminal 3-methyluridine using NaIO4 to produce dialdehydegroups for coupling with hydrazide groups of the gel and coupled to eachmicromatrix element by applying 0.5-1 nl of the activatedoligonucleotide solution (100 pmol/μl) using a specially devised robot.

Hybridization and image analysis. Probe binding was quantified bymeasuring the fluorescence conferred by the binding of fluorescentlylabeled DNA or RNA (tetramethyl rhodamine or fluorescein) to theindividual gel elements. Hybridization and washing was controlled andmonitored using a Peltier thermotable (with a working range of −5.0° C.to +60.0° C.) mounted on the stage of a custom-made epifluorescentmicroscope. The microchip was hybridized at 5° C., either overnight orfor 6 hr, in 2-5 μl of the hybridization buffer [33% formamide, 0.9 MNaCl, 1 L mM EDTA, 1% Tween-20, and 50 mM sodium phosphate (pH 7.0)] ata concentration of DNA and RNA between 0.2-2 pmol/μl. The hybridizationmixture was replaced with 5-10 μl hybridization buffer without formamideimmediately prior to microscopic observation. Exposures were in therange of 0.1-10 sec depending on the signal intensity, but weretypically around 1 sec. Fluorescence was monitored either at roomtemperature or using a range of temperatures between 5-60° C.

Conditions for the coupling of micromolecules to the acrylamide gel weredevised to rule out the possibility of liquid evaporation duringimmobilization and to ensure that covalent bonding of oligonucleotideswith the gel matrix proceeds to completion. After the microvolumes ofthe oligonucleotide solutions have been applied to all cells of thematrix, the micromatrix gel elements were swelled by condensing waterfrom the ambient air. Then the micromatrix surface was covered with athin layer of an inert nonluminescent oil, and chemical coupling of theactivated oligonucleotides to the activated polyacrylamide was carriedout to completion.

Example 6

Use of Microchip Biosensors As Diagnostic Assays

The microchip technology was successfully tested for identification ofsingle base changes in genomic DNA and RNA for reliable diagnosis ofhuman genetic diseases. A customized microchip containedoligonucleotides specific to β-thalassemia normal and abnormal β-globingenes. The hybridization with PCR-amplified DNA or RNA samples derivedfrom genomic DNA of subjects allowed unambiguous identification of amutation in a sample to be tested. Reliability of the identification wasenhanced by using simultaneous hybridization with two samples of anormal and mutated RNA stained with different fluorescence dyes andmonitoring the hybridization at different wavelengths; by simultaneouslymeasuring the melting curve for duplexes formed on a microchip, and byusing a proper set of several oligonucleotides complementary to themutated site of the DNA.

A number of the most commonly occurring P-thalassemia mutations withβ-globin gene were used in diagnostic assays with oligonucleotidemicrochip biosensors. These mutations were splice-site mutations for the1^(st), 2^(nd), 5^(th), and 6^(th) nucleotides in the first intron (IVSI) of the β-globin gene: IVS I/1 G/A (G/A=substitution of G for A), IVSI/2 T/C, IVS I/5 G/T, IVS I/5 G/C, IVS I/6 T/C, and G/A substitution inthe 26th codon (GAG) of the first exon (FIG. 6), (also known as abnormalhemoglobin E) (see Diaz-Chico et al., 1988 for terminology).

A microchip with 100H10OH20 μm gel elements (Yershov et al., 1996)contained immobilized decadeoxyribonucleotides, that is, 10-mers thatcorrespond to normal and mutant β-thalassemia alleles. These 10-mersdiscriminated mismatches less reliably than 8-mers, but were hybridizedmore efficiently than 8-mers. 10-mers were, therefore, preferred forthis assay. Table 3 shows the sequences of the allele-specificoligonucleotides immobilized on the microchips. It was expected thatmismatches within the duplexes would have a much higher destabilizationeffect than mismatches at the terminal positions (Khrapko et al., 1991);therefore the mutated bases were placed inside of the immobilizedoligonucleotides.

Single- and double-stranded PCR-amplified globin DNA fragments ofdifferent lengths and collected after a random fragmentation were testedin assays for identification of some of these mutations. However, thehybridization of RNA is preferred over DNA hybridization. RNA fragmentswere derived from PCR-amplified genomic DNA by transcription with T7 RNApolymerase (Lipshutz et al., 1995). About 100 copies of unlabeled orfluorescently labeled RNA transcripts are synthesized per DNA molecule,providing a convenient way to prepare a sufficient amount of thehybridization probes. RNA is fragmented and one fluorescent dye moleculeis introduced per fragment.

Table 3 shows the sequences of the microchip allele specific 10-mers.The oligonucleotides of microchip I are complementary to the codingstrand of DNA of the β-globin gene of patients with β-thalassemiasingle-base mutations (G/A—substitution of A for G) in the 1^(st),2^(nd), 5^(th), or 6^(th) nucleotides of the first intron (IVS I/1, 2,5, 6) of the β-globin gene and in the codon #-26 (CD-26) of the firstexon. Oligonucleotides 1-16 of microchip II correspond to the normal andIVS I/2 G/T allele. The mutated and corresponding normal bases areplaced from the 2^(nd) to the 9^(th) positions of the 10-mers from their3′-end. The mutated bases are shown in lowercase bold letters andcorresponding oligonucleotide bases in the normal allele areunderscored. The oligonucleotide synthesis and the microchipmanufacturing were described by Yershov et al. (199)6).

Microchip I was successively hybridized with RNA 75 and 133 nt longwithout fragmentation or after fragmentation (133fr, Table 5. probes 3aand 4a) and with 6 synthetic 19-mer oligodeoxyribonucleotidescorresponding to β-thalassemia mutations. The RNA and 19-mers werelabeled with TMR except for RNA probes 2a, 2b, and 6b, which werelabeled with fluorescein (Fl). The melting curves (FIGS. 1A-B, FIG. 2)were measured simultaneously for all microchip oligonucleotides at eachhybridization. These curves provided values of hybridization intensitiesat the discrimination temperature, Td. R is the ratio of thehybridization signal of a mismatched duplex (Im) to the signal of theperfect duplex (Ip) estimated at Td in parallel for all microchipoligonucleotides. R=Im/Ip. d₁₉-synthetic 19-deoxymers were complementaryto allele specific 10-mers immobilized on the microchips.

Table 4 shows the effect of the position of the allelic base within10-mers on mutation detection. Microchip II contains two sets of 10-merscorresponding to the normal and IVS I/2 T/G alleles. The microchip washybridized with the TMR-labeled normal allele 19-mer and to an RNA 75 ntlong. T^(0.1) is the temperature at which the hybridization signals fora microchip duplex drops to I/10 of its initial value at 0° C. -ΔT^(0.1)(a perfect duplex) minus T^(0.1) (the corresponding mismatched duplex.)

Fluorescently labeled RNA probes were prepared from a fragment of theβ-globin gene from the first exon (Lawn et al., 1980). PCR amplificationof a 1.76-kb fragment of the human β-globin gene mapped from nucleotides-47 to +1714 (Lawn et al., 1980) was carried out with mg genomic of DNA(Poncz et al., 1982) and 50 pmol each of the forward primer:5′-GGAGCCAGGGCTGGGCATAAAAGT-3′) (SEQ ID NO:18) (−47->-23) and thereverse primer 5′-ATTTTCCCAAGGTTTGAACTAGCTC-3′ (SEQ IDNO:19)(+1689->+1714). (FIG. 7) The amplification was carried out in aDNA thermal cycler (Gene Amp PCR System 2400, Perkin Elmer Corporationin 100 μl of a buffer containing 200 mM each of dATP, dCTP, dGTP, dTTP,2.5 mM M9Cl², 2 units of Taq DNA polymerase (BioMaster, Russia), 50 mMKCl, 10 mM Tris-Hcl, pH 9.0, and 0.1% Triton X-100. The reactionconditions were 30 cycles, with 45 sec at 95° C., 90 sec at 66° C., and120 sec at 72° C. PCR product was purified from 2% low gel/meltingtemperature agarose gel (NuSieve agarose, FMC). The 159 bp and 102-bpDNA fragments were amplified with 10 ng of the 1.75 kb DNA with threenested primers, two containing T7 promoter sequence and a common reverseprimer. The nested primers were T7-V2L-45). (5′-GGAATTCCTAATACGACTCACTATAGGGACACCATGGTGCACCTGACTCC-3′ (SEQ ID NO: 5)-44->+66); T7-V2L-103(5 ′-GAATTCCTAATACGACTCACTATAGGGAGGTGAACGTGGATGAA GTTGG-3′ (SEQ IDNO:16); +102->-123); and 5′-TCTCCTTAAACCTGTCTTGTAACC-3′ (SEQ ID NO:17)(common reverse; 153->+176). The amplification was carried out in 25cycles (15 sec at 95° C., 30 sec at 62° C., and 30 sec at 72° C.). PCRproducts were purified by QIAGEN QIAquick PCR Purification Kit. ThePCR-amplified 159 or 102 bp DNA (4-5 μg) containing T7 promoter wastranscribed with 400 units of T7 RNA polymerase (Promega) to produce 133and 75 nt long RNA in 80 μl of buffer containing 300 mM HEPES, pH 7.6,30 mM MgCl², 16 mg of BSA, 40 mM DTT, 30 units of Rnasin (Promega) and 4mM each of ATP, CTP, GTP, and UTP for 3 μL at 38° C. Deproteinization ofthe reaction mixture was carried out in 20 mM EGTA, pH 8.0, 2% SDS, andProteinase K (10 mg/ml) for 15 min at 37° C. The mixture was extractedfirst with equal volumes of phenol and then with equal volumes ofchloroform, precipitated twice by one volume of isopropyl alcohol, from0.5 M LiC10.sub.4 and dissolved on a Bio-Spin P6 column (BioRad).

Fragmentation of 10-100 μg of RNA to an average length of 20- to 40-merswas carried out in 50 μl of 0.1 M KOH for 30 min. at 40° C. Then 5 μl of1M HEPES, pH 7.6, and 15 μl of 1% HCO.sub.4 were added at 4° C. Thepellet of potassium perchlorate was removed by centrifugation and RNAwas precipitated by 10 volumes of 2% LiCIO⁴ in acetone. The RNA waswashed twice with acetone and dried for 20-30 min. at room temperature.The fragmented RNA was dephosphorylated in 50 μl of 20 mM Tris-HCl, ph8.0, 1 mM MgCl², 1 mM ZnCl², 10 units of Rnasin, 5-7 units of calfintestine phosphatase (CIP) for 1 hour at 37° C. RNA deproteinizationand purification was carried out as described herein.

For chemical fluorescence labeling of RNA the 3′-terminaldephosphorylated nucleoside was oxidized in 20 μl of 10 mM sodiumperiodate for 20 min. at room temperature. RNA was precipitated withacetone. A 10 molar excess of 10 mM TMR-hydrazine in 10% acetonitrilewas added to oxidized RNA fragments in 20 μl of 20 mM sodium acetate atpH 4.0.

The reaction mixture was incubated 30-40 min at 37° C., and thehydrazide bond between the RNA and dye was stabilized by reduction withfreshly prepared 1.5 μl of 0.2 M NacNBH³ and incubated for 30 min. atroom temperature. Then the mixture was extracted four times with watersaturated n-butanol and precipitated with acetone. Alternatively, RNAwas labeled by incorporation of fluorescein-UTP during the transcriptionwith Ambion MEGAshortscript kit according to the manual.

The hybridization of fluorescently labeled RNA (1 pmol/μl) with themicrochips was carried out at 0° C. for 18 h. In many cases, theintensities of the hybridization signals at 0° C. were similar forperfect and mismatched duplexes. The perfect and mismatched duplexes aswell as the duplexes having various GC and AT contents displayeddifferent stabilities and therefore were tested at differenttemperatures.

Table 4 summarizes the results of hybridization of the diagnosticmicrochips with 1) RNA probes derived from a number of homozygous andheterozygous β-thalassemia patients; and 2) with corresponding 19-mers.The table shows the Td for perfect duplexes formed on each microchipoligonucleotide. The relative intensities, R, of the hybridizationsignals for a different microchip oligonucleotides in Table 3 arenormalized to the signals for a perfect duplex at the Td (estimated as1.0). In most cases the ratios for mismatched duplexes are less than 0.1and close to 0. These values are low enough to allow unambiguousidentification of the homozygous and heterozygous mutations in patientsat the Td (when the hybridization signals from only perfect duplexes areobserved). The hybridization of homozygote RNA (Table 5, probes 1a, 2a,2b, and 3a) with the microchip shows the distinctive formation of aperfect duplex only with one immobilized oligonucleotide and mismatchedduplexes with all others. Two perfect duplexes were unambiguouslyidentified upon hybridization with at heterozygote RNA (Table 5, probe4a). TABLE 3 The seguence of the microchip allele specific 10-mers.Position of mutated # Allele base Sequence Location MICROCHIP I 1 IVS(N) — 5′-A TAC CAA CCT-gel (SEQ ID NO: 20) +141 2 IVS I/1 G/A 8 5′-A TACCAA tCT-gel (SEQ ID NO: 21) +141 3 IVS I/1 G/T 8 5′-A TAC CAA aCT-gel(SEQ ID NO: 22) +141 4 IVS I/2 T/A 7 5′-A TAC Cat CCT-gel (SEQ ID NO:23) +141 5 IVS I/2 T/C 7 5′-A TAC Cag CCT-gel (SEQ ID NO: 24) +141 6 IVSI/2 T/G 7 5′-A TAC Cac CCT-gel (SEQ ID NO: 25) +141 7 IVS I/5 G/A 4 5′-ATAt CAA CCT-gel (SEQ ID NO: 26) +141 8 IVS I/5 G/C 4 5′-A TAg CAACCT-gel (SEQ ID NO: 27) +141 9 IVS I/5 G/T 4 5′-A TAa CAA CCT-gel (SEQID NO: 28) +141 10 IVS I/6 T/C 3 5′-A TgC CAA CCT-gel (SEQ ID NO: 29)+141 11 CD 26 (N) — 5′-G GCC TCA CCA-gel (SEQ ID NO: 30) +125 12 CD 26G/A 6 5′-G GCC TtA CCA-gel (SEQ ID NO: 31) +125 MICROCHIP II 1 IVS (N) 95′-TGA TAC CAA C-gel (SEQ ID NO: 32) +143 2 IVS I/2 T/G 9 5′-TGA TAC CAcC-gel (SEQ ID NO: 33) +143 3 IVS (N) 8 5′-GA TAC CAA CC-gel (SEQ ID NO:34) +142 4 IVS I/2 T/G 8 5′-GA TAC CAc CC-gel (SEQ ID NO: 35) +142 5 IVS(N) 7 5′-A TAC CAA CCT-gel (SEQ ID NO: 36) +141 6 IVS I/2 T/G 7 5′-A TACCac CCT-gel (SEQ ID NO: 37) +141 7 IVS (N) 6 5′-TAC CAA CCT G-gel (SEQID NO: 38) +140 8 IVS I/2 T/G 6 5′-TAC CAc CCT G-gel (SEQ ID NO: 39)+140 9 IVS (N) 5 5′-AC CAA CCT GC-gel (SEQ ID NO: 40) +139 10 IVS I/2T/G 5 5′-AC CAc CCT GC-gel (SEQ ID NO: 41) +139 11 IVS (N) 4 5′-CCAA CCT GCC-gel (SEQ ID NO: 42) +138 12 IVS I/2 T/G 4 5′-C CAc CCTGCC-gel (SEQ ID NO: 43) +138 13 IVS (N) 3 5′-CAA CCT GCC-gel (SEQ ID NO:44) +137 14 IVS I/2 T/G 3 5′-CAc CCT GCC C-gel (SEQ ID NO: 45) +137 15IVS (N) 2 5′-AA CCT GCC CA-gel (SEQ ID NO: 46) +136 16 IVS I/2 T/G 25′-Ac CCT GCC CA-gel (SEQ ID NO: 47) +136

TABLE 4 The effect of the position of the allele base within 10-mers onmutation detection 19-mer RNA Position T_(a.1) of T_(a.1) of T_(a.1) ofT_(a.1) of allele perfect (G-A) ΔT_(a.1) perfect (G-A) ΔT_(a.1) 9 40 328 35 37 −2 8 47 32 15 49 38 11 7 42 30 12 44 41 3 6 47 28 19 49 41 8 552 38 14 50 42 8 4 54 39 15 54 44 10 3 55 46 9 59 54 5 2 52 46 6 58 53 5

TABLE 5 Identification of β-thalassemia mutations by hybridization withthe microchip Immobilized 10-mer oligonucleotide IVS I/1 I/1 I/2 I/2 I/2I/5 I/5 I/5 I/6 CD26 CD26 Hybridized (N) G/A G/T T/A T/C T/G G/A G/G G/TT/C (N) G/A Probe R at Td = Size 42° 39° 38.5° 42° 48° 45.5° 37° 44.5°40° 50° 54.5° 49° # Allele (nt) C. C. C. C. C. C. C. C. C. C. C. C. 1 aIVS (N) 75 1.00 0.04 0 0.20 0.05 0.07 0 0 0 0.04 1.0 — b IVS (N) 19^(a)1.00 0.0.9 0.07 0.002 0.03 0.01 0.03 0.03 0.07 ND 0 0 2 a IVS I/2 T/AF175 0.15 0 0 1.00 0.12 0.08 0 0 0 0 1.00 — b IVS I/2 T/A F1133 0.03 0 01.00 0 0.30 0 0 0 0 1.00 0.19 c IVS I/2 T/A 19^(a) 0.01 0 0 1.00 0.070.03 0 0 0 0 0.01 0 3 a IVS I/1 G/A 133fr 0.03 1.00 0 0.00 0 0.02 0 0 00 1.00 — b IVS I/1 G/A 19^(c) 0.01 1.00 0.01 0.01 0 0 0.01 0 0 0 0 0 4 aIVS I/1 G/A & 133fr 0.2 0.85 0 0.2 0 0.05 0 0 0 1.00 1.00 — IVS I/6 T/Cb IVS I/1 G/A 19^(c) 0.01 1.00 0.01 0.01 0 0 0.01 0 0 0 0 0 c IVS I/6T/C 19^(a) 0.1 0 0 0 0 0 0 0 0 1.0 0 0 5 a IVS I/5 G/T 19^(a) 0 0 0 0 00 0.03 0.02 1.00 0 0 0 b CD26 (N) 19^(c) 0 0 0 0 0 0 0 0 0 0 1.00 0.03 cCD26 G/A 19^(a) 0.03 0 0 0 0 0 0 0 0 0 0.04 1.00 6 a IVS (N) 75 1.000.04 0 0.20 0.05 0.07 0 0 0 0.04 1.00 — b IVS I/2 T/A F1133 0.03 0 01.00 0 0.30 0 0 0 0 1.00 —

The noticeable exceptions are oligonucleotides corresponding to IVS I/2T/A and IVS I/2 T/G mutations that show strong mismatched signals uponhybridization with non-corresponding samples of IVS (N) and IVS I/2 T/ARNA's, respectively (Table 5, 1a, 2b, 4a, 6a and 6b). The relativeintensities of these mismatched signals can be significantly decreasedby choosing the proper oligonucleotides for immobilization. It appearsthat the diagnostic assays can be carried out with RNAs 75 nucleotides(nt) long (Table 5, probes 1a, and 6a), and 133 nt long (probes 2a and6b), as well as with 133 nt long RNA fragmented to pieces 20-40 nt long(probes 3a and 4a). However, the intensities of the hybridizationsignals after fragmentation are increased by about 5 times and the timeof hybridization is decreased from several hours to a tens of minutes.

The longer RNA probes diffuse more slowly into the gel and can formstable secondary structures or aggregates. These factors interfere withtheir hybridization with rather short immobilized oligonucleotides.Thus, the fragmentation seems to be an essential step in samplepreparation, since it enhances and speeds the hybridization.

In addition to the measuring of the melting curves, the reliability ofidentification of mutations and base changes can be enhanced by the useof a multicolor fluorescence microscope (Yershov et al., 1996). For thispurpose, the tested RNA is marked by one fluorescence label and ishybridized with a microchip in the presence of a normal allele samplelabeled with a different dye. The pattern and the ratio of hybridizationmeasured with the two dyes will be similar for all microchipoligonucleotides except for those that correspond to different allelebases, i.e., mutations. Table 4 shows the results of such an experiment.The patterns of hybridization detected at two wavelengths are verysimilar.

As shown in Table 3, the immobilized 10-mers matching the mutations IVSI-2 T/G, IVS I-2 T/C, and IVS I-2 T/A are hybridized rather stronglywith some RNA probes that correspond to other alleles. Differentstructural factors in RNA could cause this hybridization. The effect ofthese factors can be minimized by placing a variable IVS I-2 base intodifferent positions of the 1 0-mers. The results of such experiments areshown in Table 4. Microchip II was successively hybridized withfragmented 75-nt-long RNA or with a synthetic DNA 19-mer, bothcorresponding to the normal allele. Microchip II contained two similarsets of eight overlapped immobilized 10-mers that are complementaryeither to a normal allele or to IVS I-2 T/G allele. The allele specificbases A for the first set and C for the second set are located in these10-mers in all internal positions from the 2d to the 9^(th). These basesform perfect A-T or mismatched A-G base pairs, respectively. Thestability of the perfect and mismatched duplexes formed on the microchipis determined as To the temperature at which the initial hybridizationsignal of the duplex is decreased to one-tenth of the originalintensity. ΔT^(0.1) corresponds to the difference in T^(0.1) between theperfect and similar mismatched duplexes. A better discrimination of theperfect and mismatched duplexes is reflected in higher values ofΔT^(0.1). The discrimination efficiency (ΔT) was lower for hybridizedRNAs than for the 19-mers. The discrimination was surprisingly low,ΔT=−2′ and 3° C., when the allelic bases were placed at the 9^(th) or7^(th) position, respectively, of the immobilized oligonucleotides. Itappears that secondary structures and the presence of similar sequencesin other regions of the RNA causes this lowering. These effects can bepartly predicted from the sequence of the region that is searched formutations. However, it is impossible to reach a high discrimination(ΔT=8-11° C.) when allele bases are placed in other positions, forexample the 8, 6, 5, or 4 positions.

The hybridization of RNA transcripts of PCR-amplified DNA witholigonucleotide microchips allows the reliable identification of basechanges and discrimination of homozygous and heterozygous β-thalassemiamutations in the genomic DNA of patients.

RNA transcribed from PCR-amplified DNA provides an easier method forpreparing a sufficient amount of labeled, single-stranded samples thanthe use of DNA prepared by PCR amplification. RNA can be fragmented andone fluorescent dye molecule can be introduced per fragment.

Example 7

Use of a Customized Microchip Biosensor to Detect Gene Expression

Gene expression is one of the central themes in modern molecularbiology. DNA from well studied genetical sources has already beensystematically sequenced. For these sequences hybridization proceduresare successfully used to estimate a level of differential geneexpression. The results of this estimation are useful for understandingfundamental mechanisms of development biology, embryology and treatmentof genetic and infectious diseases.

To determine whether oligonucleotide microchips are useful to identifygene expression, microchip biosensor hybridization was carried out withssDNA fragments isolated from six different genes:

-   -   205 b fragment from glyceraldehyde 3-phosphate dehydrogenase        (G3PDH);    -   281 b fragment from human transferrin receptor (HTR);    -   224 b fragment from human β.sub.2-microglobulin (B2M);    -   545 b fragment from human interleukin-1 receptor (ILIR);    -   188 b fragment from human NF-kB (p50);    -   224 b fragment from human interferon γ receptor (IGR).

A customized microchip, containing immobilized 60 b oligonucleotides,having at the 3′-terminal position 3-methyluridine residues,corresponding to five house-keeping genes (G3PDH, HTR, B2M, ILIR andNF-kb(p50)) (CLONTECH catalog 94/95 “Tools for the Molecular Biologist”,pp. 90-93) were produced for hybridization experiments withcomplementary ssDNA fragments. Each oligonucleitide was applied at twopositions on the microchip in a 1:10 ratio of amount (0.3 pmol:0.03 pmoleach). ssDNA fragments complementary to immobilized oligonucleotideswere synthesized by asymmetric PCR amplification (using only one primer)with fluorescently labeled nucleotide triphosphates (FUORscript T7,Fluorescein-Labeling In Vitro Transcription Kit). Moreover, thePCR-primer bore a biotin tag that was utilized for following isolationof synthesized ssDNA fragments with avidin carried on a column (Sambrooket al. “Molecular Cloning” 2d edit., p. 12.14). FIG. 8 demonstrateshybridization on the microchip. Intensity of fluorescence in each spotdepends on the amount of immobilized oligonucleotide and on the lengthof the DNA fragment in the spot. For hybridization, 10 μl of Buffer A(50% formamide, 10% dextran sulfate, 1% SDS, 50 mM sodium phosphate atpH 7.4, 750 mM sodium chloride, 5 mM sodium EDTA) containing ssDNA witha concentration of 0.5 pmol/μl (approximately 0.05 μg/μl) was incubatedfor about 6-12 h at room temperature, washed briefly with H₂O andanalyzed with a fluorescent microscope. Before rehybridization themicrochip was treated in Buffer B (50% formamide, 1% Tween 20) for 30min. at 50° C. to completely remove hybridized ssDNA.

These results indicate that concentration of fluorescently labeled ssDNAmay be decreased up to 100 fold. Hybridization with individual ssDNAfragments indicates high specificity of studied oligonucleotides. Therewas no cross-hybridization detected between different tested DNAs andimmobilized oligonucleotides. None of the oligonucleotides demonstrateda signal when hybridized with non-specific DNA (e.g. probe IGR). Thisdifferentiates “expression” of non-expressed genes from expression ofhousekeeping genes. Genes that are not expressed in a particular cell ortissue, may actually be picked up in conventional screening proceduresas having a low expression, while other genes being expressed in allcells (housekeeping genes) will also be picked up as having low tomoderate expression. The housekeeping genes are actually beingexpressed. In this example a difference in signal is detectable so thatlow level expression could be unambiguously distinguished from low levelbackground.

The procedure detects expression of genes of high and middle expressionlevel. To determine low level gene expression selective RT-PCRamplification is preferred.

Example 8

Use of a Customized Microchip Biosensor of the Present Invention toDetect HLA Polymorphisms

A difficult problem of genotype recognition arises in studying differenthaplotypes (alleles) of genes encoding Human Leucocyte Antigens (HLA) inregions of histocompatibility genes. The HLA locus (class I and class IIgenes) is responsible for histocompatibility of tissue transplantation.The need for allele identification is encountered also in variousmedical and biological tasks involving HLA class II genes. There aremany clinical data showing strong association between HLA genotype andsusceptibility to some disorders, for example some alleles DQA1/DQB1 areclearly related to IDDM (Insulin-Dependent Diabetes Mellitus), malaria,autoimmune diseases, such as rheumatoid arthritis and pemphigusvulgaris—a skin disease which causes severe blistering. The high levelof polymorphism of HLA has been shown to be useful for identification ofindividuals determining the group of risk for some diseases. HLA typingis particularly crucial for matching donors for transplants. It is alsoproposed for infertility work-ups.

In this aspect, the present invention provides a method which allows anarray of immobilized 8-12 bp-long oligonucleotides to form amoligonucleotide microchip thereby facilitating identification of HLADQA1 allies.

An algorithm has been designed and special computer programs have beenconstructed which allow the analysis of the nucleotide sequences of allalleles of various HLA subloci. Forming an optimized set of oligonucleotides provides high reliability of detection of homo- andheterozygotes for the HLA alleles.

A customized microchip, containing an array of eighteen PAA-gelimmobilized (1 pmol of each) short oligonucleotides has been producedfor hybridization with fluorescently labelled complementary HLA DQA1 DNAor RNA probe for allele identification. 18 decamers were loaded on thechip in the following order, from left to right: TABLE 6 First 1 2 3 4g⁴ - control (upper) row oligo Second: 5 6 7 8 g³ - control oligo Third:9 10 11 12 13 Fourth: 14 15 16 17 18

The sequence of the oligos used was as shown in FIG. 9.

The oligonucleotides immobilized on the microchip are complementary tothe sense strand of different alleles of DQA1 DNA and some controloligos. A microchip with 20 oligonucleotides was manufactured forpartial identification of 15 different alleles in the HLA DQA1 region.PCR was used to prepare 229 bp (starting from condon 12 to condon 87)DNA fragments of the polymorphic second exon of the DQA1 gene from humangenomic DNA. Nested primers were used 2DQAA1P-A:5′-a t ggt gta aac ttgtac cag t (SEQ ID NO:73); and 2DQAAMP-B:5′tt ggyt agc agrc ggt aga gtt g(SEQ ID NO:74). Nested PCR primers were: T7-2DQAAMP-A and primer B. Thefirst primer containing the promoter for T7 RNA polymerase and PCRproduct were used for in vitro transcription. RNA probes were identicalto the coding DNA strand. RNA was fragmented, labeled with fluoresceinand used for hybridization with the microchip. Hybridization conditionswere as follows: overnight incubation at 5° C. in 1M NaCl, 1 mM EDTA, 5mM Na-phosphate, pH 7.0, 1% Tween 20. The temperature was then increasedstepwise at 10° C. intervals, and fluorescence measurements were takenat each step. BUFFER WAS NOT CHANGED.

FIG. 10 shows the hybridization results and presents schematically theHLA DQA1-chip for allele identification. In FIG. 10 three diagonallyplaced oligos (11-0101/0104 allele specific; 6-specific for 0101, 01021,01022, 0103, 00104; 17-correspond to all alleles, except 0502) gave apositive hybridization signal, and are observed as three diagonallyplaced bright fluorescence spots. The probes were identified as the 0101or 0104 allele (both alleles are identical in the second exon). Allother oligos yielded much weaker fluorescence signals compared withthose described above, because none of them contain sequencescomplementary to alleles 0101 and 0104. On the other hand any alleledifferent from 0101 or 0104, reveals another set of hybridizationsignals.

The brightest fluorescent squares on the chip were: Oligo#4 which is03011 or 0302 specific; oligo#8 is Taq polymerase-specific artifacts;oligo#18-belong to alleles 0101-05011; oligo 11-0101, 0104 allelespecific; 17-corresponds to all alleles, except 0502; g4 is afluorescent control oligo; # 13-mismatch to # 18. All other chipelements showed significantly less intensive fluorescence. The genotypeidentified by these probes has a 0101/0104-0302/03011 heterozygote.

Example 9

Use of a Customized Microchip Biosensor to Detect the Lyme DiseaseSpirochetes

Bacteria belonging to the species Barretia burgdorferi and relatedspecies of tick-borne spirochetes are capable of causing human andveterinary disease. Nucleic acid probes are available to detect bacteriacausing Lyme disease. These bacteria cannot be identified by standardmicrobiological methods, although immimological tests are available.

Using the methods of the present invention, oligonucleotides areprepared according to Weisburg (1995) and added to a microniatrixdesigned for use in detecting Lyme disease in a clinical sample.

Example 10

Use of a Customized Microchip Biosensor to Detect Salmonella In FoodSamples

Salmonella presence is detected most commonly by preparing culturesaccording to standard microbiological laboratory procedures, and testingthe cultures for morphological and biochemical characteristics. Afterabout 48 hours after collection of a sample testing begins and takesseveral days to complete.

However, RNA and DNA probes for Salmonella testing are available. Usingthe methods of the present invention, oligonucleotides are preparedaccording to Lane et al. (1996) incorporated herein by reference andadded to a microchip designed for use in detecting Salmonella in foodsamples by distinguishing rRNA of Salmonella from non-Salmonella.

1-4. (canceled) 5-10. (canceled)
 11. (canceled)
 12. A method using non-equilibrium melting curves to detect mismatches between a nitrogen base sequence on a microchip and a nitrogen base sequence to be tested, said method comprising: (a) generating the non-equilibrium melting curves by simultaneously monitoring hybridization between the nitrogen base sequence that matches or mismatches with sequences on the microchip at a series of temperatures; (b) selecting the temperature at which maximum discrimination occurs between the match and the mismatch; and (c) determining the degree of mismatch of the nitrogen base sequence to be tested at the selected temperature.
 13. The method of claim 12, wherein the selected temperature is that temperature at which the signal intensity of a mismatched sequence is at least one tenth of the signal intensity of a matched sequence.
 14. (canceled)
 15. (canceled) 16-17. (canceled) 18-31. (canceled) 